Intracellular bacterial pathogens constitute a serious threat to human health and antibiotic drugs are largely used to prevent and/or cure bacterial infection. These chemical compounds mainly act by targeting the bacterial machinery and, unfortunately, cases of resistance among pathogens are being selected in the process, leaving us with no effective treatment against these deviant pathogenic strains. These serious aspects of drug treatment point to the necessity of developing not only new drugs to counter resistance, but also new conceptual approaches in order to identify new treatments. In this application, we propose to develop a new approach to drug discovery by developing high throughput screening (HTS) assays for imaging intracellular pathogen infection in mammalian cells. To this end, we model the infection process using Listeria monocytogenes, a cytosolic bacterial pathogen and HeLa cells, a human epithelial cell line. We first propose to develop and optimize an HTS assay for imaging and quantifying Listeria infection in mammalian cells by automated fluorescence microscopy and computer assisted image analysis. We will test the reproducibility and robustness of our assay by screening a library of 20,000 compounds available at the Yale Center for Genomics and Proteomics. Next, in order to determine the degree of specificity of the identified candidates, we propose to develop secondary assays to test for cell toxicity, bacterial killing and potential activity on other pathogens upon infection. We will also develop secondary HTS assays to further characterize Listeria-specific candidates and precisely determine which step of Listeria infection is affected by the activity of the corresponding candidates. Finally, we propose to utilize the optimized primary and secondary assays and conduct parallel chemical and genetic screens to identify bacterial and host factors affecting the Listeria infection process. Our ultimate objective is to conduct comparative analyses of the phenotypes observed in chemical and genetic screens to potentially identify bioactive molecules and their corresponding target genes. We believe that the use of the proposed assays in various approaches, such as chemical and genetic screens, will provide a comprehensive understanding of the biological process under investigation, the interaction of an intracellular pathogen and its host cell. Beyond their biomedical implications, these studies may also further our understanding of the native cellular functions of the targeted host factors.